RESUMEN
Rubisco activity is highly regulated and frequently limits carbon assimilation in crop plants. In the chloroplast, various metabolites can inhibit or modulate Rubisco activity by binding to its catalytic or allosteric sites, but this regulation is complex and still poorly understood. Using rice Rubisco, we characterised the impact of various chloroplast metabolites which could interact with Rubisco and modulate its activity, including photorespiratory intermediates, carbohydrates, amino acids; as well as specific sugar-phosphates known to inhibit Rubisco activity - CABP (2-carboxy-d-arabinitol 1,5-bisphosphate) and CA1P (2-carboxy-d-arabinitol 1-phosphate) through in vitro enzymatic assays and molecular docking analysis. Most metabolites did not directly affect Rubisco in vitro activity under both saturating and limiting concentrations of Rubisco substrates, CO2 and RuBP (ribulose-1,5-bisphosphate). As expected, Rubisco activity was strongly inhibited in the presence of CABP and CA1P. High physiologically relevant concentrations of the carboxylation product 3-PGA (3-phosphoglyceric acid) decreased Rubisco activity by up to 30%. High concentrations of the photosynthetically derived hexose phosphates fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) slightly reduced Rubisco activity under limiting CO2 and RuBP concentrations. Biochemical measurements of the apparent Vmax and Km for CO2 and RuBP (at atmospheric O2 concentration) and docking interactions analysis suggest that CABP/CA1P and 3-PGA inhibit Rubisco activity by binding tightly and loosely, respectively, to its catalytic sites (i.e. competing with the substrate RuBP). These findings will aid the design and biochemical modelling of new strategies to improve the regulation of Rubisco activity and enhance the efficiency and sustainability of carbon assimilation in rice.
Asunto(s)
Cloroplastos , Simulación del Acoplamiento Molecular , Oryza , Ribulosa-Bifosfato Carboxilasa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/química , Cloroplastos/metabolismo , Cloroplastos/enzimología , Oryza/metabolismo , Oryza/enzimología , Fotosíntesis , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Dióxido de Carbono/metabolismo , Ribulosafosfatos/metabolismo , Fructosafosfatos/metabolismoRESUMEN
The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROOâ¢) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROOâ¢, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROOâ¢. Enzyme mixtures (1:1:1 ratio) were exposed to ROO⢠produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated â¼175 µM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (â¼100 µM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.
Asunto(s)
Escherichia coli , Glucosafosfato Deshidrogenasa , NADP , Oxidación-Reducción , Vía de Pentosa Fosfato , Fosfogluconato Deshidrogenasa , Glucosafosfato Deshidrogenasa/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , NADP/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Ribulosafosfatos/metabolismo , Glucosa-6-Fosfato/metabolismo , Peróxidos/metabolismo , Hidrolasas de Éster CarboxílicoRESUMEN
Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.
Asunto(s)
Anhidrasas Carbónicas , Cianobacterias , Ribulosa-Bifosfato Carboxilasa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/genética , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/química , Cianobacterias/metabolismo , Cianobacterias/genética , Cianobacterias/enzimología , Regulación Alostérica , Filogenia , Ribulosafosfatos/metabolismo , Modelos Moleculares , Multimerización de Proteína , Dióxido de Carbono/metabolismo , Especificidad por Sustrato , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/químicaRESUMEN
Oxygenic photosynthesis evolved in cyanobacteria, primary producers of striking ecological importance. Like plants, cyanobacteria use the Calvin-Benson-Bassham cycle for CO2 fixation, fuelled by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). In a competitive reaction this enzyme also fixes O2 which makes it rather ineffective. To mitigate this problem, cyanobacteria evolved a CO2 concentrating mechanism (CCM) to pool CO2 in the vicinity of RuBisCO. However, the regulation of these carbon (C) assimilatory systems is understood only partially. Using the model Synechocystis sp. PCC 6803 we characterized an essential LysR-type transcriptional regulator encoded by gene sll0998. Transcript profiling of a knockdown mutant revealed diminished expression of several genes involved in C acquisition, including rbcLXS, sbtA and ccmKL encoding RuBisCO and parts of the CCM, respectively. We demonstrate that the Sll0998 protein binds the rbcL promoter and acts as a RuBisCO regulator (RbcR). We propose ATTA(G/A)-N5 -(C/T)TAAT as the binding motif consensus. Our data validate RbcR as a regulator of inorganic C assimilation and define the regulon controlled by it. Biological CO2 fixation can sustain efforts to reduce its atmospheric concentrations and is fundamental for the light-driven production of chemicals directly from CO2 . Information about the involved regulatory and physiological processes is crucial to engineer cyanobacterial cell factories.
Asunto(s)
Ribulosa-Bifosfato Carboxilasa , Synechocystis , Dióxido de Carbono/metabolismo , Oxigenasas/metabolismo , Fotosíntesis/genética , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosafosfatos , Synechocystis/metabolismoRESUMEN
Cerium oxide nanoparticles (CeO2 NPs) and zinc oxide nanoparticles (ZnO NPs) are emerging pollutants that are likely to occur in the contemporary environment. So far, their combined effects on terrestrial plants have not been thoroughly investigated. Obviously, this subject is a challenge for modern ecotoxicology. In this study, Pisum sativum L. plants were exposed to either CeO2 NPs or ZnO NPs alone, or mixtures of these nano-oxides (at two concentrations: 100 and 200 mg/L). The plants were cultivated in hydroponic system for twelve days. The combined effect of NPs was proved by 1D ANOVA augmented by Tukey's post hoc test at p = 0.95. It affected all major plant growth and photosynthesis parameters. Additionally, HR-CS AAS and ICP-OES were used to determine concentrations of Cu, Mn, Fe, Mg, Ca, K, Zn, and Ce in roots and shoots. Treatment of the pea plants with the NPs, either alone or in combination affected the homeostasis of these metals in the plants. CeO2 NPs stimulated the photosynthesis rate, while ZnO NPs prompted stomatal and biochemical limitations. In the mixed ZnO and CeO2 treatments, the latter effects were decreased by CeO2 NPs. These results indicate that free radicals scavenging properties of CeO2 NPs mitigate the toxicity symptoms induced in the plants by ZnO NPs.
Asunto(s)
Cerio/farmacología , Nanopartículas del Metal/química , Nutrientes , Fotosíntesis , Pisum sativum/fisiología , Óxido de Zinc/farmacología , Cerio/metabolismo , Pisum sativum/efectos de los fármacos , Pisum sativum/crecimiento & desarrollo , Fotosíntesis/efectos de los fármacos , Pigmentos Biológicos/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , Estomas de Plantas/efectos de los fármacos , Estomas de Plantas/fisiología , Transpiración de Plantas/efectos de los fármacos , Ribulosafosfatos/metabolismo , Zinc/metabolismoRESUMEN
Ribulose 1,5-bisphosphate (RuBP) undergoes enolization to initiate fixation of atmospheric carbon dioxide in the plant carbon cycle. The known model assumes the binding of RuBP to the Rubisco active site with the subsequent formation of 2,3-enediol (2,3,4-trihydroxypent-2-ene-1,5-diyl diphosphate). In the present study, it is assumed that 1,2-enol (2,3,4-trihydroxypent-1-ene-1,5-diyl diphosphate) can be formed in the enolization step to initiate the carboxylation reaction. We have used Kohn-Sham density functional theory on WB97X-D3/Def2-TZVP levels to compare the reaction barriers in the two ways. We considered the pathways of carboxylation of 1/2-ene (mono/di)ol via the C1 and C2 carbons without taking into account the binding of RuBP to the magnesium ion. Calculations of Gibbs free energies confirm the equal probability of the formation of 2,3-enediol and 1,2-enol. Quantum-chemical modeling of enolization and carboxylation reactions supports the important role of the bridging water molecule and diphosphate groups, which provide proton transfer and lower reaction barriers. The results show that carbon dioxide fixation can occur without a magnesium ion, and binding with C1 can have a lower barrier (~12 kcal/mol) than with C2 (~23 kcal/mol).
Asunto(s)
Dióxido de Carbono , Modelos Químicos , Ribulosafosfatos/química , Algoritmos , Dióxido de Carbono/química , Catálisis , Estructura MolecularRESUMEN
BACKGROUND: Clusters of sex-specific loci are predicted to shape the boundaries of the M/m sex-determination locus of the dengue vector mosquito Aedes aegypti, but the identities of these genes are not known. Identification and characterization of these loci could promote a better understanding of mosquito sex chromosome evolution and lead to the elucidation of new strategies for male mosquito sex separation, a requirement for several emerging mosquito population control strategies that are dependent on the mass rearing and release of male mosquitoes. This investigation revealed that the methylthioribulose-1-phosphate dehydratase (MtnB) gene, which resides adjacent to the M/m locus and encodes an evolutionarily conserved component of the methionine salvage pathway, is required for survival of female larvae. RESULTS: Larval consumption of Saccharomyces cerevisiae (yeast) strains engineered to express interfering RNA corresponding to MtnB resulted in target gene silencing and significant female death, yet had no impact on A. aegypti male survival or fitness. Integration of the yeast larvicides into mass culturing protocols permitted scaled production of fit adult male mosquitoes. Moreover, silencing MtnB orthologs in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus revealed a conserved female-specific larval requirement for MtnB among different species of mosquitoes. CONCLUSIONS: The results of this investigation, which may have important implications for the study of mosquito sex chromosome evolution, indicate that silencing MtnB can facilitate sex separation in multiple species of disease vector insects.
Asunto(s)
Aedes/enzimología , Anopheles/enzimología , Culex/enzimología , Hidroliasas/metabolismo , Proteínas de Insectos/metabolismo , Aedes/genética , Aedes/crecimiento & desarrollo , Animales , Anopheles/genética , Anopheles/crecimiento & desarrollo , Culex/genética , Culex/crecimiento & desarrollo , Femenino , Hidroliasas/genética , Proteínas de Insectos/genética , Larva/enzimología , Larva/genética , Larva/crecimiento & desarrollo , Masculino , Ribulosafosfatos/metabolismoRESUMEN
Giardia lamblia, due to the habitat in which it develops, requires a continuous supply of intermediate compounds that allow it to survive in the host. The pentose phosphate pathway (PPP) provides essential molecules such as NADPH and ribulose-5-phosphate during the oxidative phase of the pathway. One of the key enzymes during this stage is 6-phosphogluconate dehydrogenase (6 PGDH) for generating NADPH. Given the relevance of the enzyme, in the present work, the 6pgdh gene from G. lamblia was amplified and cloned to produce the recombinant protein (Gl-6 PGDH) and characterize it functionally and structurally after the purification of Gl-6 PGDH by affinity chromatography. The results of the characterization showed that the protein has a molecular mass of 54 kDa, with an optimal pH of 7.0 and a temperature of 36-42 °C. The kinetic parameters of Gl-6 PGDH were Km = 49.2 and 139.9 µM (for NADP+ and 6-PG, respectively), Vmax =26.27 µmol*min-1*mg-1, and Kcat = 24.0 s-1. Finally, computational modeling studies were performed to obtain a structural visualization of the Gl-6 PGDH protein. The generation of the model and the characterization assays will allow us to expand our knowledge for future studies of the function of the protein in the metabolism of the parasite.
Asunto(s)
Giardia lamblia/enzimología , Gluconatos/química , NADP/química , Fosfogluconato Deshidrogenasa/química , Proteínas Protozoarias/química , Ribulosafosfatos/química , Secuencias de Aminoácidos , Sitios de Unión , Clonación Molecular/métodos , Expresión Génica , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/enzimología , Giardia lamblia/genética , Gluconatos/metabolismo , Humanos , Cinética , Modelos Moleculares , NADP/metabolismo , Vía de Pentosa Fosfato/genética , Fosfogluconato Deshidrogenasa/genética , Fosfogluconato Deshidrogenasa/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulosafosfatos/metabolismo , Homología Estructural de Proteína , Especificidad por Sustrato , TermodinámicaRESUMEN
The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.
Asunto(s)
Azúcares de Nucleósido Difosfato/biosíntesis , Nucleotidiltransferasas/química , Oxidorreductasas/química , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Pared Celular/metabolismo , Cristalografía por Rayos X , Espectrometría de Masas/métodos , Modelos Moleculares , Mutación , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Oxidorreductasas/metabolismo , Pentosafosfatos/metabolismo , Multimerización de Proteína , Ribulosafosfatos/metabolismo , Staphylococcus aureus/citología , Staphylococcus aureus/enzimologíaRESUMEN
Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M-1cm-1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1-3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.
Asunto(s)
Isomerasas Aldosa-Cetosa/análisis , Dicroismo Circular/métodos , Pruebas de Enzimas/métodos , Proteínas Bacterianas/metabolismo , Catálisis , Francisella tularensis/metabolismo , Lipopolisacáridos/metabolismo , Pentosafosfatos/metabolismo , Ribulosafosfatos/análisis , Ribulosafosfatos/metabolismo , Especificidad por Sustrato , Azúcares Ácidos/metabolismo , Fosfatos de Azúcar/metabolismoRESUMEN
The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.
Asunto(s)
Proteínas Bacterianas/metabolismo , Gluconacetobacter/enzimología , Gluconatos/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Ribulosafosfatos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Gluconacetobacter/genética , Gluconatos/química , Humanos , Modelos Químicos , Modelos Moleculares , Estructura Molecular , NAD/metabolismo , NADP/metabolismo , Fosfogluconato Deshidrogenasa/clasificación , Fosfogluconato Deshidrogenasa/genética , Filogenia , Dominios Proteicos , Multimerización de Proteína , Ribulosafosfatos/química , Homología de Secuencia de AminoácidoRESUMEN
Methanol is a biotechnologically promising substitute for food and feed substrates since it can be produced renewably from electricity, water and CO2. Although progress has been made towards establishing Escherichia coli as a platform organism for methanol conversion via the energy efficient ribulose monophosphate (RuMP) cycle, engineering strains that rely solely on methanol as a carbon source remains challenging. Here, we apply flux balance analysis to comprehensively identify methanol-dependent strains with high potential for adaptive laboratory evolution. We further investigate two out of 1200 candidate strains, one with a deletion of fructose-1,6-bisphosphatase (fbp) and another with triosephosphate isomerase (tpiA) deleted. In contrast to previous reported methanol-dependent strains, both feature a complete RuMP cycle and incorporate methanol to a high degree, with up to 31 and 99% fractional incorporation into RuMP cycle metabolites. These strains represent ideal starting points for evolution towards a fully methylotrophic lifestyle.
Asunto(s)
Escherichia coli/metabolismo , Metanol/metabolismo , Ribulosafosfatos/metabolismo , Proteínas Bacterianas , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/metabolismo , Ingeniería Metabólica , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
The paper briefly describes the evolution of the key enzyme of photosynthesis, RuBisCO. Before the emergence of the reaction of carbon dioxide assimilation via photosynthesis, this protein was involved in the methionine metabolism chain. Possibly, for this reason, the carboxylation reaction catalyzed by enzyme proceeds very slowly. In addition to carboxylation, RuBisCO can simultaneously oxidize ribulose bisphosphate, a substrate to which the fixed CO2 is attached. This, in turn, also reduces the effectiveness of photosynthesis. In this regard, the literature discusses various options for increasing plant productivity by creating new forms of RuBisCO or fundamentally different pathways of carbon dioxide assimilation. In this work, we propose a modification of the carboxylation reaction that makes it possible to avoid photorespiration and thus increase the efficiency of photosynthesis.
Asunto(s)
Bacillus subtilis/metabolismo , Dióxido de Carbono/química , Fotosíntesis , Ribulosa-Bifosfato Carboxilasa/química , Ribulosafosfatos/química , Carbono/química , Catálisis , Cinética , Oxígeno/química , FotoquímicaRESUMEN
Methanol is a sustainable substrate for biotechnology. In addition to natural methylotrophs, metabolic engineering has gained attention for transfer of methylotrophy. Here, we engineered Corynebacterium glutamicum for methanol-dependent growth with a sugar co-substrate. Heterologous expression of genes for methanol dehydrogenase from Bacillus methanolicus and of ribulose monophosphate pathway genes for hexulose phosphate synthase and isomerase from Bacillus subtilis enabled methanol-dependent growth of mutants carrying one of two independent metabolic cut-offs, i.e., either lacking ribose-5-phosphate isomerase or ribulose-5-phosphate epimerase. Whole genome sequencing of strains selected by adaptive laboratory evolution (ALE) for faster methanol-dependent growth was performed. Subsequently, three mutations were identified that caused improved methanol-dependent growth by (1) increased plasmid copy numbers, (2) enhanced riboflavin supply and (3) reduced formation of the methionine-analogue O-methyl-homoserine in the methanethiol pathway. Our findings serve as a foundation for the engineering of C. glutamicum to unleash the full potential of methanol as a carbon source in biotechnological processes.
Asunto(s)
Corynebacterium glutamicum/genética , Evolución Molecular Dirigida/métodos , Metanol/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Microbiología Industrial/métodos , Ingeniería Metabólica/métodos , Riboflavina/metabolismo , Ribulosafosfatos/metabolismo , TransgenesRESUMEN
Studies on Glucose-6-phosphate (G6P)/phosphate translocator isoforms GPT1 and GPT2 reported the viability of Arabidopsis (Arabidopsis thaliana) gpt2 mutants, whereas heterozygous gpt1 mutants exhibited a variety of defects during fertilization/seed set, indicating that GPT1 is essential for this process. Among other functions, GPT1 was shown to be important for pollen and embryo-sac development. Because our previous work on the irreversible part of the oxidative pentose phosphate pathway (OPPP) revealed comparable effects, we investigated whether GPT1 may dually localize to plastids and peroxisomes. In reporter fusions, GPT2 localized to plastids, but GPT1 also localized to the endoplasmic reticulum (ER) and around peroxisomes. GPT1 contacted two oxidoreductases and also peroxins that mediate import of peroxisomal membrane proteins from the ER, hinting at dual localization. Reconstitution in yeast (Saccharomyces cerevisiae) proteoliposomes revealed that GPT1 preferentially exchanges G6P for ribulose-5-phosphate (Ru5P). Complementation analyses of heterozygous +/gpt1 plants demonstrated that GPT2 is unable to compensate for GPT1 in plastids, whereas GPT1 without the transit peptide (enforcing ER/peroxisomal localization) increased gpt1 transmission significantly. Because OPPP activity in peroxisomes is essential for fertilization, and immunoblot analyses hinted at the presence of unprocessed GPT1-specific bands, our findings suggest that GPT1 is indispensable in both plastids and peroxisomes. Together with its G6P-Ru5P exchange preference, GPT1 appears to play a role distinct from that of GPT2 due to dual targeting.
Asunto(s)
Antiportadores/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Peroxisomas/metabolismo , Plastidios/metabolismo , Alelos , Aminoácidos/metabolismo , Antiportadores/química , Proteínas de Arabidopsis/química , Citosol/metabolismo , Fertilización , Glucosa-6-Fosfato/metabolismo , Modelos Biológicos , Proteínas de Transporte de Monosacáridos/química , Óvulo Vegetal/metabolismo , Oxidación-Reducción , Filogenia , Dominios Proteicos , Multimerización de Proteína , Transporte de Proteínas , Ribulosafosfatos/metabolismo , Semillas/metabolismo , Estrés FisiológicoRESUMEN
The Calvin-Benson-Bassham (CBB) cycle is responsible for CO2 assimilation and carbohydrate production in oxyphototrophs. Phosphoribulokinase (PRK) is an essential enzyme of the CBB cycle in photosynthesis, catalyzing ATP-dependent conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-bisphosphate. The oxyphototrophic PRK is redox-regulated and can be further regulated by reversible association with both glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and oxidized chloroplast protein CP12. The resulting GAPDH/CP12/PRK complex is central in the regulation of the CBB cycle; however, the PRK-CP12 interface in the recently reported cyanobacterial GAPDH/CP12/PRK structure was not well resolved, and the detailed binding mode of PRK with ATP and Ru5P remains undetermined, as only apo-form structures of PRK are currently available. Here, we report the crystal structures of cyanobacterial (Synechococcus elongatus) PRK in complex with ADP and glucose-6-phosphate and of the Arabidopsis (Arabidopsis thaliana) GAPDH/CP12/PRK complex, providing detailed information regarding the active site of PRK and the key elements essential for PRK-CP12 interaction. Our structural and biochemical results together reveal that the ATP binding site is disrupted in the oxidized PRK, whereas the Ru5P binding site is occupied by oxidized CP12 in the GAPDH/CP12/PRK complex. This structure-function study greatly advances the understanding of the reaction mechanism of PRK and the subtle regulations of redox signaling for the CBB cycle.
Asunto(s)
Arabidopsis/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fotosíntesis , Synechococcus/enzimología , Adenosina Trifosfato/metabolismo , Proteínas de Arabidopsis/metabolismo , Biocatálisis , Dominio Catalítico , Ligandos , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Estructura Secundaria de Proteína , Ribulosafosfatos/metabolismo , Homología Estructural de ProteínaRESUMEN
The oxidative pentose phosphate pathway (oxiPPP) contributes to cell metabolism through not only the production of metabolic intermediates and reductive NADPH but also inhibition of LKB1-AMPK signaling by ribulose-5-phosphate (Ru-5-P), the product of the third oxiPPP enzyme 6-phosphogluconate dehydrogenase (6PGD). However, we found that knockdown of glucose-6-phosphate dehydrogenase (G6PD), the first oxiPPP enzyme, did not affect AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or 6-phosphogluconolactonase (PGLS), the second oxiPPP enzyme, reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6-phosphogluconolactone level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. Furthermore, γ-6-phosphogluconolactone, an oxiPPP byproduct with unknown function generated through intramolecular rearrangement of δ-6-phosphogluconolactone, the only substrate of PGLS, bound to Src and enhanced PP2A recruitment. Together, oxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Gluconatos/metabolismo , Neoplasias/enzimología , Proteína Fosfatasa 2/metabolismo , Células A549 , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Proliferación Celular , Activación Enzimática , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Células HEK293 , Células HT29 , Humanos , Células K562 , Células MCF-7 , Ratones Desnudos , Neoplasias/genética , Neoplasias/patología , Células PC-3 , Vía de Pentosa Fosfato , Unión Proteica , Proteína Fosfatasa 2/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ribulosafosfatos/metabolismo , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Carga Tumoral , Familia-src Quinasas/metabolismoRESUMEN
Food production in green crops is severely limited by low activity and poor specificity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in natural photosynthesis (NPS). This work presents a scientific solution to overcome this problem by immobilizing RuBisCO into a microfluidic reactor, which demonstrates a continuous production of glucose precursor at 13.8 µmol g-1 RuBisCO min-1 from CO2 and ribulose-1,5-bisphosphate. Experiments show that the RuBisCO immobilization significantly enhances enzyme stabilities (7.2 folds in storage stability, 6.7 folds in thermal stability), and also improves the reusability (90.4% activity retained after 5 cycles of reuse and 78.5% after 10 cycles). This work mimics the NPS pathway with scalable microreactors for continuous synthesis of glucose precursor using very small amount of RuBisCO. Although still far from industrial production, this work demonstrates artificial synthesis of basic food materials by replicating the light-independent reactions of NPS, which may hold the key to food crisis relief and future space colonization.
Asunto(s)
Enzimas Inmovilizadas/metabolismo , Glucosa/biosíntesis , Microfluídica/métodos , Fotosíntesis , Ribulosa-Bifosfato Carboxilasa/metabolismo , Dióxido de Carbono/metabolismo , Productos Agrícolas/metabolismo , Estabilidad de Enzimas , Glucosa/química , Hojas de la Planta/metabolismo , Reproducibilidad de los Resultados , Ribulosafosfatos/metabolismo , TemperaturaRESUMEN
BACKGROUND: Efficient xylose fermentation still demands knowledge regarding xylose catabolism. In this study, metabolic flux analysis (MFA) and metabolomics were used to improve our understanding of xylose metabolism. Thus, a stoichiometric model was constructed to simulate the intracellular carbon flux and used to validate the metabolome data collected within xylose catabolic pathways of non-Saccharomyces xylose utilizing yeasts. RESULTS: A metabolic flux model was constructed using xylose fermentation data from yeasts Scheffersomyces stipitis, Spathaspora arborariae, and Spathaspora passalidarum. In total, 39 intracellular metabolic reactions rates were utilized validating the measurements of 11 intracellular metabolites, acquired by mass spectrometry. Among them, 80% of total metabolites were confirmed with a correlation above 90% when compared to the stoichiometric model. Among the intracellular metabolites, fructose-6-phosphate, glucose-6-phosphate, ribulose-5-phosphate, and malate are validated in the three studied yeasts. However, the metabolites phosphoenolpyruvate and pyruvate could not be confirmed in any yeast. Finally, the three yeasts had the metabolic fluxes from xylose to ethanol compared. Xylose catabolism occurs at twice-higher flux rates in S. stipitis than S. passalidarum and S. arborariae. Besides, S. passalidarum present 1.5 times high flux rate in the xylose reductase reaction NADH-dependent than other two yeasts. CONCLUSIONS: This study demonstrated a novel strategy for metabolome data validation and brought insights about naturally xylose-fermenting yeasts. S. stipitis and S. passalidarum showed respectively three and twice higher flux rates of XR with NADH cofactor, reducing the xylitol production when compared to S. arborariae. Besides then, the higher flux rates directed to pentose phosphate pathway (PPP) and glycolysis pathways resulted in better ethanol production in S. stipitis and S. passalidarum when compared to S. arborariae.
Asunto(s)
Fermentación , Análisis de Flujos Metabólicos/métodos , Metaboloma , Metabolómica/métodos , Saccharomycetales/metabolismo , Fructosafosfatos/metabolismo , Glucosa-6-Fosfato/metabolismo , Glucólisis , Malatos/metabolismo , Espectrometría de Masas/métodos , Modelos Biológicos , Vía de Pentosa Fosfato , Ribulosafosfatos/metabolismo , Saccharomycetales/clasificación , Levaduras/clasificación , Levaduras/metabolismoRESUMEN
Large numbers of genes essential for embryogenesis in Arabidopsis encode enzymes of plastidial metabolism. Disruption of many of these genes results in embryo arrest at the globular stage of development. However, the cause of lethality is obscure. We examined the role of the plastidial oxidative pentose phosphate pathway (OPPP) in embryo development. In nonphotosynthetic plastids the OPPP produces reductant and metabolic intermediates for central biosynthetic processes. Embryos with defects in various steps in the oxidative part of the OPPP had cell division defects and arrested at the globular stage, revealing an absolute requirement for the production via these steps of ribulose-5-phosphate. In the nonoxidative part of the OPPP, ribulose-5-phosphate is converted to ribose-5-phosphate (R5P)-required for purine nucleotide and histidine synthesis-and subsequently to erythrose-4-phosphate, which is required for synthesis of aromatic amino acids. We show that embryo development through the globular stage specifically requires synthesis of R5P rather than erythrose-4-phosphate. Either a failure to convert ribulose-5-phosphate to R5P or a block in purine nucleotide biosynthesis beyond R5P perturbs normal patterning of the embryo, disrupts endosperm development, and causes early developmental arrest. We suggest that seed abortion in mutants unable to synthesize R5P via the oxidative part of the OPPP stems from a lack of substrate for synthesis of purine nucleotides, and hence nucleic acids. Our results show that the plastidial OPPP is essential for normal developmental progression as well as for growth in the embryo.